Discovery of a Potent and Highly Selective Dipeptidyl Peptidase IV and Carbonic Anhydrase Inhibitor as “Antidiabesity” Agents Based on Repurposing and Morphing of WB-4101

The management of patients with type 2 diabetes mellitus (T2DM) is shifting from cardio-centric to weight-centric or, even better, adipose-centric treatments. Considering the downsides of multidrug therapies and the relevance of dipeptidyl peptidase IV (DPP IV) and carbonic anhydrases (CAs II and V) in T2DM and in the weight loss, we report a new class of multitarget ligands targeting the mentioned enzymes. We started from the known α1-AR inhibitor WB-4101, which was progressively modified through a tailored morphing strategy to optimize the potency of DPP IV and CAs while losing the adrenergic activity. The obtained compound 12 shows a satisfactory DPP IV inhibition with a good selectivity CA profile (DPP IV IC50: 0.0490 μM; CA II Ki 0.2615 μM; CA VA Ki 0.0941 μM; CA VB Ki 0.0428 μM). Furthermore, its DPP IV inhibitory activity in Caco-2 and its acceptable pre-ADME/Tox profile indicate it as a lead compound in this novel class of multitarget ligands.


■ INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a chronic condition characterized by the dysregulation of carbohydrate, lipid, and protein metabolism and results from impaired insulin secretion, insulin resistance, or a combination of both. Of the three major types of diabetes, T2DM is far more common (accounting for more than 90% of all cases) than either type 1 diabetes mellitus (T1DM) or gestational diabetes. 1 Globally, 6.28% of the world's population is affected by T2DM. Developed regions such as Western Europe show higher prevalence rates that continue to increase despite public health measures. The distribution of T2DM generally matches the socioeconomic development even though the burden of suffering due to diabetes is rapidly increasing in lower-income countries. 2 Thus, T2DM is recognized as a global public health concern, which directly impacts human life and health expenditures. With regard to the cost of diabetes care, it is 3.2 times greater than the average per capita healthcare expenditure, even reaching 9.4 times in the presence of comorbidities. As a matter of fact, over time, diabetes can damage the heart, blood vessels, eyes, kidneys, and nerves. The damages occur more frequently in patients affected by T2DM since symptoms are less marked than those revealed to T1DM; thus, the disease may be diagnosed several years after the onset. Moreover, T2DM incidence has increased in lockstep with the obesity pandemic during the last half-century. 3 In fact, the numbers of individuals with T2DM parallel the numbers of adults, with obesity generating a worldwide dual epidemic, which is an important public health issue. 4 The detrimental health effects of diabetes and obesity are well known so much that they are described by the term "diabesity".
For these reasons and considering that the two conditions share key pathophysiological mechanisms, the management of patients with T2DM is shifting from a cardio-centric goal to a new weight-centric, or, even better, adipose-centric treatment goal. 5 Since diabetes is characterized by a complex physiopathology, pharmacological monotherapies have proven ineffective in controlling blood glucose levels and other comorbidities. Therefore, therapeutic treatment is frequently done through drug combinations, which operate with different mechanisms of action 6 but with potential drug−drug interaction risks. Moreover, a complex combinatorial regimen is one of the leading causes of nonadherence to therapeutic recommendations 7 and this represents a serious concern since compliance is a conditio sine qua non for improving outcomes for patients with diabetes.
In a state of metabolic disturbance, like the one affecting both diabetic and obese patients, multitarget drugs that concomitantly normalize glycemia and inhibit the progression of comorbidities could be a useful option for the management of these conditions. Given the relevance of dipeptidyl peptidase IV (DPP IV) and carbonic anhydrase isoforms II and V (CAs II and V) roles in the pathology of T2DM as well as in the weight loss, multitarget ligands able to modulate these enzymes could represent a promising therapeutic approach for antidiabesity treatment. DPP IV plays an important role in maintaining glucose homeostasis since it is responsible for the inactivation of the incretin hormones, namely, the glucosedependent insulinotropic polypeptide hormone (GIP) and the glucagon-like peptide 1 (GLP-1). 8 These endocrine hormones are released from the gut in response to intraluminal carbohydrates, and they are implicated in numerous desirable pancreatic actions, including insulin secretion and gene expression stimulation, increasing β-cell survival, enhancing β-cell glucose sensitivity, and reducing glucagon production. 9,10 Human carbonic anhydrases (hCAs) are ubiquitous zincmetalloenzymes, which act as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate and protons. They are involved in many conditions either physiological or pathological such as electrolyte secretion and biosynthetic reactions like gluconeogenesis, lipogenesis, and ureagenesis. In particular, isoforms II and V play a significant role in metabolic processes such as gluconeogenesis. 11 CA II is the most physiologically relevant isoform and it has been found in several organs/tissues. Several studies demonstrated that CA II interacts with a variety of membrane-bound carriers to regulate the cytoplasmic pH, such as the sodium/hydrogen exchanger (NHE1) 12 or the sodium bicarbonate cotransporter (NBC1). 13 Recent investigation shows that NHE1/CA II metabolon complex is exacerbated in diabetic cardiomyopathy of ob −/− mice, which may lead to perturbation of intracellular pH and Na + and Ca 2+ concentrations, contributing to cardiovascular anomalies. Moreover, the enhanced NHE1/ CA II metabolon activity was correlated with an increased CA II expression in hypertrophic and functionally impaired ob −/− mice hearts. 14 Evidence proves also that CA II is overexpressed in diabetic ischemic human myocardium.
CA V is, among all of the isoforms, the only one located in the mitochondria. There are two mitochondrial CAs with different tissue distributions and they are usually referred to as CA VA and CA VB. These isoforms influence many physiological processes, like CO 2 transport, bone resorption, gluconeogenesis, production of body fluids, lipogenesis, ureagenesis, and de novo synthesis of HCO 3 − within the mitochondrial compartment. 15 Mitochondrial HCO 3 − is essential for pyruvate carboxylase in the gluconeogenic or in lipogenic pathways and for carbamoyl phosphate synthetase I in the ureagenesis process in the liver. This is because HCO 3 − cannot permeate the inner mitochondria membrane and no bicarbonate transporter (SLC4A family) is located on this membrane. Therefore, HCO 3 − supplied by CA VA/VB is thus critical for pyruvate carboxylase to convert pyruvate to oxaloacetate in the mitochondrion. Then, the tricarboxylate transporter transports the oxaloacetate to the cytosol, where it is implicated in the synthesis of fatty acids (lipogenesis). 16 Furthermore, CA V is also most likely implicated in the glucose-induced secretion of insulin. 17 There are at least two possible mechanisms by which mitochondrial CAs could participate in the regulation of insulin secretion. In detail, as already mentioned, CA V provides HCO 3 − for pyruvate carboxylase, which is abundantly expressed in the mitochondrial islet cells, and it is important in the pyruvate−malate shuttle, which provides NADPH for normal β-cell functions, like glucose-induced secretion of insulin, as demonstrated by a study of MacDonald et al. 18 Furthermore, the control of mitochondrial calcium concentrations is a second way through which CA V may be connected to insulin secretion. As observed by Kennedy et al., calcium ions have a fundamental role in the energy requirements for the exocytosis of insulin from β-cell. 19 In addition, there is clear evidence that the inhibition of both the isoforms VA and VB leads to considerable weight loss. 11,20,21 Given these considerations, DPP IV and CA (II and V) can be considered good targets to treat concomitantly T2DM and obesity, which are often linked. Therein, a new class of multitarget ligands addressed on DPP IV and CA (II and V) is reported. The derivates have been designed by combining a repurposing strategy and a subsequent morphing process. Indeed, drug repurposing has been recognized as a good tool to speed up the drug discovery and drug development pipeline, while morphing allows molecular properties to be progressively improved through rational drug design.
Among the various amenable α 1 -AR antagonists, we chose as template WB-4101 (2-[(2,6-dimethoxyphenoxyethyl)aminomethyl]-1,4-benzodioxane ( Figure 1) for several reasons: (a) preliminary docking simulations showed that it is conveniently harbored within the DPP IV cavity where it elicits clear interactions with some of the key residues of the S1 and S2 subsites (see Figure 2); (b) our laboratory has accumulated significant experience in the synthesis of this compound and of its derivatives; 28−30 (c) we learned through previous studies that were also confirmed by the literature how to reduce or abrogate the α 1 -AR affinity by substituting the para position of the phenoxy ring. 31,32 Clearly, we do not expect that this repurposed molecule could be active per se on the targets and, however, this should not be actually desirable due to its strong effect on the adrenergic receptors. Thus, we decided to modify WB-4101 as little as possible to gain inhibition toward DPP IV and CAs while losing the adrenergic affinity. These tailored modifications have been introduced by a progressive morphing strategy that provides the major advantage of scanning the specific contribution of each modification besides allowing the modulation of the activity toward the old and new targets. 33,34 Specifically, the introduced modifications regarded two portions of the structure of WB-4101: the already mentioned para position of the 2,6-dimethoxyphenoxy ring and the secondary amine function.
At first, the para position of the 2,6-dimethoxyphenoxy ring was substituted by the sulfonamide group, which should have a beneficial effect on both CAs and DPP IV while reducing adrenergic affinity. This modification was supported by preliminary docking simulations on the resolved CA II structure (see Figure S1, Supporting Information), which showed that the sulfonamide group is able to properly chelate Zn ++ and that the WB-4101 analogue is conveniently accommodated within the CA II cavity where it mostly stabilizes hydrophobic contacts. Similarly, docking simulations on DPP IV revealed that the para-sulfonamide group does not affect the already shown binding mode of WB-4101 even though the introduced sulfonamide moiety fails to properly contact Arg358 (see Figure S2, Supporting Information). We also designed compounds characterized by reversed sulfonamide moieties to elongate the para-substituents in an attempt to reach Arg358. Subsequently, besides maintaining the sulfonamide residue, we decided to transform the secondary amine present in WB-4101 into a primary amine, also designing the corresponding azide derivatives. Such a modification besides being suggested by the observation that gliptins generally include a primary amino group or a nitrile residue has been driven by the continuous feedback coming from biological assays. Therefore, different WB-4101 derivatives have been designed and synthesized. The compounds described therein can be divided into three sets (Scheme 1). Compounds belonging to the first set differ very little from WB-4101 since the two ortho methoxy groups were removed and the aromatic ring was decorated by different para-sulfonamide moieties. The substitution in the para position was chosen to abrogate or at least diminish the alpha-adrenoceptor activity, as already demonstrated. The second set covers the compounds that feature the azide group instead of the secondary amine and one, two, or no methoxy group on the phenoxy ring substituted at the para position with a primary sulfonamide. The third set includes the analogues of the second set that are characterized by a primary amine in lieu of the azide function. These modifications were inspired by the promising docking investigation and common features shared by drugs already available on the market.
Chemistry. According to the structure, the designed compounds can be divided into three sets: the first one includes the derivatives characterized by a secondary amine; the second one comprises the ligands endowed by an azide group; and the third covers the analogues of the second set, which carries a primary amine instead of the azide moiety.
Phenol was reacted with 1-bromo-2-chloroethane to give 2chloroethoxybenzene (15), which underwent an acylation in the para position with chlorosulfonic acid. Afterward, the  nucleophilic substitution with different amines gave the corresponding sulfonamide moiety (17a−e). Through a subsequent substitution with sodium azide followed by a reduction using hydrazine, synthons 19a−e were obtained. The synthesis of building block 25 is reported in Scheme 3.
The synthetic strategy started from the protection of the amino group of commercially available p-aminophenol with ditert-butyl dicarbonate (20). Subsequently, the hydroxyl moiety underwent an hydroxyalkoxylation to provide tert-butyl (4hydroxyethoxy)phenyl)carbamate (21), which is also shared by the synthetic strategy planned to obtain 30a and 30b. The  synthetic approach used to obtain 25 continued using methanol hydrochloride to deprotect the amino group, which was mesylated with methanesulfonyl chloride as well as the hydroxy group to provide 23. Then, the nucleophilic substitution with sodium azide and the eventual reduction with hydrazine gave amine 25.
The synthetic route to obtain 30a and 30b started from the shared synthon 21 whose hydroxyl function was mesylated (26) and subsequently substituted by sodium azide (27).
The deprotection of the amine function (28) was achieved by treatment with methanol hydrochloride. Then, through the reaction with ethane-or isobutene-sulfonyl chloride, the sulfonamide synthons (29a−b) were obtained. The azide function was then reduced with hydrazine giving amine 30a−b, which were condensed with 2-mesyloxymethyl-1,4-benzodioxane giving the free secondary amine derivatives, which gave compounds 7 and 8 after the treatment with diethyl ether hydrochloride.
The preparation of compounds 9−14 shared a common key 38 for which the synthetic route is illustrated in Scheme 4. The treatment of 1,4-benzodioxan-2-carboxylic acid with N,Odimethylhydroxylamine, preceded by the conversion into the corresponding acyl chloride, led to the obtainment of the Weinreb amide (31), which was reduced and transformed into Scheme 3. Synthesis of the Intermediates 25 and 30a,b a 32. The aldehydic function underwent a Reformatsky reaction with ethyl bromoacetate, accomplishing the β-hydroxy ester (33). The reduction of the ester moiety afforded the primary alcohol (34), which was protected with trityl chloride (35). After the Mitsunobu reaction, which produced the azide derivative (36), the deprotection of trityl ether was achieved via treatment with Amberlyst 15 (37).
Hence, the hydroxyl moiety was mesylated to achieve 38. The synthesis of intermediate 40 shown in Scheme 5 started from the commercially available sulfonamide whose amine function was converted into a hydroxy one (39) via the Scheme 5. Synthesis of Intermediates 40, 43, and 46 a a Sandmeyer reaction. Consequently, 40 was prepared from 4hydroxybenzenesulfonamide by the protection of the sulfonamide moiety as N-sulfonylformamidine. The synthetic strategy to achieve scaffold 43 is shown in Scheme 5. Regioselective monobromination of 4-hydroxybenzenesulfonamide gave compound 41, which was treated with sodium methoxide and CuI at reflux to obtain 42. Consequently, 43 was achieved via protection of the sulfonamide group with N,N-dimethylformamide dimethyl acetal. The obtainment of scaffold 46, shown in Scheme 5, started from 4-hydroxybenzenesulfonamide (39), which underwent ortho-dibromination. The treatment of intermediate 44 with sodium methoxide and CuI at reflux led to the obtainment of 45. The protection of the sulfonamide moiety as N-sulfonylformamidine accomplished scaffold 46.
DPP IV Inhibition. The DPP IV inhibition activities of the entire sets of compounds were carried out on purified recombinant human DPP IV using conditions previously optimized. 35 Sitagliptin and WB-4101 were included in the sets for comparison. The inhibition data are listed in Table 1. The group of these multitarget ligands, which carries a secondary amine function (1−8), does not show a noticeable inhibitory activity for DPP IV. Indeed, the data expressed as micromolar concentration reported in Table 1 highlight the absence of activity toward this enzyme for all derivatives except compounds 6 and 8, even if their profile is still not satisfactory. This trend can be partially explained by the weaker Glu−dyad interaction established by the secondary amine. Moreover, compounds 5−8 that feature reversed sulfonamide moieties appended at the para position of the phenoxy ring are inactive or show weak inhibition as well. Compounds belonging to both the second and the third sets (9−14), which are characterized by the presence of an azide or a primary amine moiety, show a different trend of inhibition. The biological   results display that derivative compounds 9 and 10 weakly inhibit DPP IV, while 11 has a strong activity on DPP IV. The docking results of this compound exhibit a distinctive binding mode with the azide moiety close to the Ser630 ( Figure 3A). Compounds 12 and 14 possess a nanomolar potency comparable to that shown by the commercially available DPP IV inhibitor sitagliptin. This strong activity could be explained by the docking poses ( Figure 3B); indeed, they assume a conformation that stabilizes all of the key interactions with the binding site like the primary amine group near the two key residues, namely, Glu205 and Glu206.
However, if we compare the binding pose of all of these derivatives, 12, 13, and 14, we observe an almost perfect overlay between the three molecular structures, making the biological results of 13 quite unexpected. In addition, all inhibitors were further stabilized by π−π stacking interactions with Tyr662 and Phe357. In the case of compound 14, the decoration of the para benzenesulfonamide with two methoxy moieties reinforces the interaction with Phe357 ( Figure 3B), enhancing the inhibitor activity. Furthermore, as assumed, by considering all compounds 9−14, a significant role seems to be played by the substituent on the phenoxy ring since the decoration by the methoxy groups results in a general positive contribution to the inhibition activity.
CA Inhibition. The CA inhibition profiles of all of the compounds (1−14) were evaluated on several common isoforms, namely, hCA I, II, IV, V (A and B), and IX. Acetazolamide (AAZ) was used as positive control, while sitagliptin and WB-4101 were included in the sets for comparison. The choice of these isoforms was based on their sequence homology to better evaluate the selectivity profile. An applied photophysics stopped-flow instrument was used for assaying the CA-catalyzed CO 2 hydration activity. 36 The results are listed in Table 1 expressed as micromolar concentrations.
The primary sulfonamide moiety of compound 1 shows strong inhibitor activity on the physiologically dominant isoform hCA II (K i 7.2 nM) with comparable potency to that of the AAZ (K i of 12.1 nM). Moreover, this compound has also shown a good selectivity profile against hCA II. Derivatives of the second and third groups show affinity comparable to that of compound 1, thus indicating that the added primary amine, as well as the azide function, does not affect the interaction with hCAs. Compounds 10 and 11 effectively and selectively inhibit the mitochondrial isoform hCA VA with inhibition constants ranging in the nanomolar range between 77.5 and 89.6 nM. Both these compounds bear the azide moiety and have at least one methoxy group on the phenoxy ring. Moreover, dimethoxy derivatives in the second and third generations are generally less efficacious on hCAs in comparison to the compounds of the same wave, leading to the conclusion that this substitution is disadvantageous on the CAs while being quite influential on the DPP IV inhibition. The reasons behind this behavior might be found in the different electronic properties of the sulfonamide moiety, which is now bound to a more electron-rich system, which influences its chelating capability. 37 Furthermore, the increased steric hindrance due to the two methoxy groups can significantly influence the approach to the protein binding site. 38 Among this generation, compound 12, which bears a primary amine moiety and has no decoration on the phenoxy ring, targets hCA II, VA, and VB with selectivity over the cytosolic isozymes (selectivity ratio hCA I/II of 7.24 and hCA IX/II of 3.68) all in the low nanomolar ranges (36.1, 94.1, and 42.8, respectively). α 1a -AR Binding Assay of Active Compounds. The most active compounds that combined DPP IV and CAs (II and/or V) inhibitory activity, namely, 11 and 12, which, however, derived from repurposing and morphing an adrenergic ligand, were tested on human cloned α 1a -AR expressed in HEK293 cells. Since WB-4101 is a potent α 1 antagonist, this adrenoreceptor subtype was chosen to assess the loss of adrenergic affinity.
As highlighted by the data reported in Figure S3 (see the Supporting Information), the compounds did not show activity on α 1a . More in detail, the data reported the absence of the binding affinity of compound 11 toward α 1a -AR, while derivative 12 exhibits poor affinity (pK i of 6.61) for the same subtype. Such results confirm that the incorporation of a moiety in the para position of the phenoxy ring of WB-4101 abrogates or at least lowers the adrenergic affinity. ADME Prediction. Pharmacokinetics, druglikeness, and medicinal chemistry friendliness have been evaluated by Swiss ADME. 39 At a first glance from the bioavailability radar plot (Figure 4), the properties of compound 11 ( Figure 4A) are not entirely comprised of the pink area but comparable to drugs containing warheads.
In addition, compound 11 shows very poor gastrointestinal absorption. Conversely, compound 12 shows a high gastrointestinal absorption and it gratifyingly falls into the pink area ( Figure 4B) indicating the suitable physicochemical space for oral bioavailability. The ilog P (log Po/w) is 0.00, the Xlog P3 (log Po/w) is 2.22, the Wlog P (log Po/w) is −1.36, the Mlog P (log Po/w) is −3.12, the SILICOS-IT (log Po/w) is 0.93, and the consensus log Po/w is −0.97. Considering the log P values, overall compound 12 has a poor lipophilic character. Furthermore, not least of all, compound 12 shows an enhanced druglikeness profile compared to WB-4101 (see Supporting Information Table S1).
Regarding water solubility, the results coming from the three methods applied by the web tool assess that the screened compounds are water-soluble. Moreover, compound 12 shows high gastrointestinal absorption and does not permeate the blood−brain barrier. In addition, compound 12 is not a P-gp substrate; thus, the excretion will not be an issue and it does not inhibit anyone of the isoenzymes, namely, CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, considered by Swiss ADME, thus excluding the chance of accumulation, drug−drug interaction, and subsequent toxicity. Additionally, compound 12 reports no PAINS alerts. The MetaClass 40 approach suggests a reasonable metabolic stability for compound 12 since it identifies only two possible reactions, namely, redox reactions on carbon atoms and acetylation of the primary amino group. Based on the overall data collected, compound 12 was selected for additional assays to frame it as a lead compound of this novel class of multitarget ligands. As a first step, compound 12 was tested in Caco-2 cells that express high levels of DPP IV on their cellular membranes. 41 Experiments were performed using sitagliptin as a reference compound. Notably, considering that, as indicated in Table 1, the sitagliptin is almost five times more active than compound 12; the concentrations used for the cell-based evaluation were 20.0 nM and 1.0 μM for sitagliptin, and 100.0 nM and 5.0 μM for compound 12. Figure 5 indicates that sitagliptin inhibited cellular DPP IV activity by 44.93 ± 1.21 and 79.69 ± 3.23% at 20 nM and 1.0 μM, respectively, vs untreated cells, whereas compound 12 inhibited cellular DPP IV activity by 60.65 ± 4.92 and 71.86 ± 4.27% at 100.0 nM and 5.0 μM, respectively, vs untreated cells ( Figure 5). Statistical analysis indicated that at a higher concentration no significant differences were observed between compound 12 and sitagliptin. The encouraging results, afforded by Caco-2 cells, confirm the activity on DPP IV even in a more complex environment.
In Vitro Pre-ADME/Tox Profiling. Solubility. The solubility of compound 12 was determined in phosphate-buffered saline at a pH of 7.4 through a suitable liquid chromatography and tandem mass spectrometry (LC-MS/MS) method, as described in the Experimental Section. Compound 12 shows a soluble concentration of 224 μM, which satisfies one of the prerequisites for good bioavailability to encourage oral dosing as the possible route of administration.
Hepatic Microsome Stability. To predict the metabolic fate as well as the susceptibility of compound 12 to phase I metabolism following the in vivo administration, it was incubated with human (Sekisui XenoTech, LLC), CD-1 mouse (BioIVT), and Sprague-Dawley (SD) rat liver microsomes (Corning), as described in the Experimental Section. As reported in Figure 6, compound 12 exhibited modest stability in mouse and rat microsomes, with 27.5 and 16.7% of the compound remaining, respectively, after 120 min of incubation. In human liver microsomes, compound 12 exhibited very high stability, with 72.6% remaining after 120 min of incubation.
Permeability. The ability of compound 12 to be uptaken by Caco-2 cells was assessed, exploiting its natural behavior to emit fluorescence at 313 nm after excitation at 250 nm using a fluorescent plate reader. Notably, Caco-2 cells were treated with compound 12 at 1.0 μM or vehicle for 15, 30, and 60 min. Fluorescence signals emitted at 313 nm by the compound localized at the intracellular level as a function of time have been normalized for the total amount of cells stained using Janus Green (OD 595 nm); therefore, the relative fluorescence unit (RFU) 313/595 nm after 15, 30, and 60 min of incubation was calculated. The RFU 313/595 nm of the blank samples, which represents the cellular background, was subtracted from each normalized fluorescence signal. Results showed that compound 12 successfully permeates the human intestinal cells as a function of time. In particular, compound 12 is absorbed by Caco-2 cells up to 2874 ± 383.1, 4562 ± 993.2, and 6250 ± 1163RFU after 15, 30, and 60 min, respectively ( Figure 7).
Cytotoxicity. The cytotoxicity of the active compound 12 was determined. Cell viability was assessed by the quantitative colorimetric method of the MTT reduction on Caco-2 and HepG2 cells. As reported in Figure S4 (see the Supporting Information), both the cell lines maintain their metabolic activity when treated with compound 12.

■ CONCLUSIONS
T2DM is steadily growing in high-income countries as well as in low-income countries. Moreover, the numbers of patients who suffer from T2DM parallel those affected by obesity, giving reasons for reframing the treatment of T2DM. Thus, new therapeutic approaches able to target diabesity are needed. Given the relevance of the DPP IV/CA (II and V) roles in both T2DM and obesity, multitarget ligands able to modulate these enzymes could represent a novel and promising therapeutic approach. Repositioning of WB-4101 and morphing its structure have been the strategies applied to obtain potent and selective multitarget ligands against DPP IV and CAs (isoforms II and V). In this context, rational multitarget optimization strategies have relied on computational models and systematic exploration of the structure−activity relationship (SAR) on each individual target. The designed sets of molecules, which result from the morphing of the phenoxy ring and the variation of the secondary amine function of WB-4101, can be roughly divided into three generations. All of the compounds have been synthesized and investigated for their inhibitory activity on the selected targets, namely, CA II, V, DPP IV, and off-targets (CA I, IV, and IX). The first generation of derivatives possesses no multitarget modulatory efficacy, despite a remarkable CA II inhibitor potency shown by compound 1. Compound 11 from the second set and compounds 13 and 14, which belong to the third group, inhibit DPP IV at nanomolar concentrations, which are comparable to sitagliptin. In addition, they show an interesting CA inhibitor profile, enriched by satisfactory selectivity. On the basis of the structure−activity relationships, a primary amine function closely connected to a rigid substructure, which is, in turn, joined to a p-hydroxybenzenesulfonamide portion, results in a crucial feature, to adequately modulate the selected enzymes.
The satisfactory DPP IV inhibition and the selectivity CA profile together with the acceptable ADME prediction confirmed by in vitro pre-ADME/Tox profiling indicate compound 12 as the potential lead of this novel class of multitarget ligands even if the design and synthesis of other new morphed derivatives of WB-4101 will help to further understand the key recognition features of the target enzymes. Albeit the characteristics, the issue of fixed activity as a multitarget ligand must be carefully considered to maintain the modulation capacity over the selected targets.
The design strategy applied in this study emphasizes that repurposing analyses can also be successfully used as a preliminary step to identify druglike starting scaffolds, which are amenable to further improvements by finely tuned and progressive modifications.
This new class of molecules paves the way for the development of a promising and reframed therapeutic approach for the treatment of T2DM and obesity. ■ EXPERIMENTAL SECTION Chemistry. All chemicals and solvents were purchased from Merck KGaA, Darmstadt, Germany, and TCI and used as commercially distributed. All purifications were performed by flash chromatography using prepacked Biotage Sfar columns or silica gel (particle size 40−63 μm, Merck) on an Isolera (Biotage, Uppsala, Sweden) apparatus. Thin-layer chromatography (TLC) analyses were performed on aluminum plates precoated with silica gel 60 matrix with a fluorescent indicator and visualized in a TLC UV cabinet followed by an appropriate staining reagent. The content of solvents in eluent mixtures is given as v/v percentage. R f values are given for guidance. 1 H NMR (300 MHz) and 13    . The purity of all tested compounds was higher than 95% and was determined on a Phenomenex Gemini C 18 column (250 mm, 4.6 mm, 5 μm, Phenomenex). High-resolution mass spectra were acquired with an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Milan, Italy) equipped with an ESI source: full MS spectra were acquired in profile mode by the FT analyzer in a scan range of m/z 120−800, using a resolution of 30,000 full width at half-maximum (FWHM) at m/z 400. The purity for all the compounds was >95%.

4-(2-Chloroethoxy)benzenesulfonamide (17a).
Gaseous ammonia was bubbled in an ice-cooled solution of 4-(2-chloroethoxy)benzenesulfonyl chloride 16 (850 mg, 3.33 mmol) in 20 mL of dichloromethane until saturation. The reaction mixture was stirred for 45 min. Afterward, ammonium chloride was removed by filtration, and dichloromethane was evaporated under vacuum to obtain the pure product 17a as a white solid (730 mg, 3.12 mmol). Mp 115°C.  N-dimethylbenzenesulfonamide 18d (290 mg, 1.07 mmol) in methanol (9 mL). Then, the mixture was refluxed, under stirring, until TLC indicated the disappearance of the starting material. Afterward, the catalyst was removed by filtration, and methanol was evaporated in vacuo. The resulting crude was dissolved in ethyl acetate, and the organic phase was extracted with a 10% aqueous solution of HCl. After being basified to pH 10 with a 10 M solution of KOH, the aqueous layer was further extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo, affording 261 mg (1.07 mmol) of the pure product 19d as a pale yellow solid. TLC (dichloromethane/ methanol 90:10 + 1% aq. NH 3 ); R f = 0.22. Mp 69°C. 1
N-(4-(2-Aminoethoxy)phenyl)-2-methylpropan-1-sulfonamide (30b). Palladium(II) oxide (28.16 mg, 0.23 mmol) and hydrazine hydrate (1.11 mL, 22.80 mmol) were added to a solution of N-(4-(2azidoethoxy)phenyl)ethanesulfonamide 29b (679 mg, 2.28 mmol) in methanol (18 mL). Then, the mixture was refluxed, under stirring, until TLC indicated the disappearance of the starting material. Afterward, the catalyst was removed by filtration, and methanol was evaporated in vacuo, affording 621 mg (2.28 mmol) of the pure product 30b as an orange oil. TLC (dichloromethane/methanol 90:10 + 0.5% aq. NH 3 ); R f = 0.32. 1  N-Methoxy-N-methyl-1,4-benzodioxan-2-carboxamide (31). Thionyl chloride (3.26 mL, 44.74 mmol) was added dropwise to an ice-cooled solution of 1,4-benzodioxan-2-carboxylic acid (4.03 g, 22.37) in 40 mL of dichloromethane. The mixture was stirred at reflux until NMR indicated the disappearance of the starting material. Afterward, the solvent was evaporated in vacuo, and the crude was dissolved in 30 mL of dichloromethane and cooled down to 0°C. Then, N-dimethylhydroxylamine hydrochloride (3.27 g, 33.56 mmol) was added in small amounts to the mixture and the reaction was stirred until TLC indicated the disappearance of the starting material. Afterward, the reaction was diluted with additional dichloromethane, washed three times with 10% aqueous solution of NaHCO 3 and brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated in vacuo, providing the pure product 31 as a yellow solid (4.69 g, 21.00 mmol). 1  1,4-Benzodioxan-2-carboxyaldehyde (32). Under a nitrogen atmosphere, a solution of N-methoxy-N-methyl-1,4-benzodioxan-2carboxamide 31 (2 g, 8.46 mmol) in 32 mL of tetrahydrofuran was added dropwise to a suspension of 425 mg of LiAlH 4 (11.20 mmol) in tetrahydrofuran (8 mL) and allowed to cool down to −20°C. The reaction mixture was stirred at the same temperature until TLC indicated the disappearance of the starting material. Afterward, the excess of LiAlH 4 was quenched by slowly adding 10% aqueous solution of HCl and diluted with dichloromethane. After the separation of the phases, the aqueous layer was further extracted with dichloromethane. The reunited organic phases were washed with brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated in vacuo, providing the pure product 32 as a yellow oil (1. . The neat product quickly degraded at room temperature but was stable for several days if dissolved in dichloromethane (ca. 20% w/v).
Ethyl 3-(1,4-Benzodioxan)-3-hydroxypropanoate (33). Under a nitrogen atmosphere, 1.21 g of zinc (18.52 mmol) and 209 mg of TBDMSiCl (1.39 mmol) were suspended in 12 mL of tetrahydrofuran. The mixture was stirred at 50°C for 40 min. Then, a solution of 1.52 g of 1,4-benzodioxan-2-carboxyaldehyde 32 (9.26 mmol) and 1.54 mL of ethyl bromoacetate (13.89 mmol) in 24 mL of tetrahydrofuran was added dropwise to the suspension. The reaction mixture was stirred at 50°C until TLC indicated the disappearance of the starting material. Afterward, the solution was diluted with ethyl acetate and the organic layer was sequentially washed with 10% aqueous solution of HCl, 10% aqueous solution of NaHCO 3 , saturated solution of Na 2 SO 3 , and finally with brine. The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was evaporated in vacuo, providing the pure product 33 as a pale yellow oil ( (34). Under a nitrogen atmosphere, a solution of ethyl 3-(1,4-benzodioxan)-3-hydroxypropanoate 33 (1.59 g, 6.30 mmol) in 20 mL of tetrahydrofuran was added dropwise to a suspension of 298 mg of LiAlH 4 (7.87 mmol) in tetrahydrofuran (5 mL) and allowed to cool down to −10°C. The reaction mixture was stirred at the same temperature until TLC indicated the disappearance of the starting material. Afterward, the excess of LiAlH 4 was quenched by slowly adding a 10% aqueous solution of HCl and diluted with dichloromethane. After the separation of the phases, the aqueous layer was further extracted with dichloromethane. The reunited organic phases were washed with brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated in vacuo, affording a yellow oil. Flash chromatography (cyclohexane/ethyl acetate 1:1) was performed to obtain 630 mg 1-(1,4-Benzodioxan)-3-trityloxy-1-propanol (35). Triethylamine (0.50 mL, 3.52 mmol) was added dropwise to an ice-cooled solution of 673 mg of 1-(1,4-benzodioxan)-1,3-propanediol 34 (3.20 mmol) and was dissolved in 10 mL of dichloromethane. The reaction mixture was warmed to room temperature, and then a solution of trityl chloride (981 g, 3.52 mmol) in 15 mL of dichloromethane was added dropwise to the solution. Then, the mixture was stirred, until TLC indicated the disappearance of the starting material. Afterward, the mixture was diluted with dichloromethane, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo, affording 1. 1-Azido-1-(1,4-benzodioxan)-3-trityloxypropane (36). Nitrogen was bubbled in a solution of 1-(1,4-benzodioxan)-3-trityloxy-1propanol 35 (1.23 g, 2.83 mmol) in 22 mL of dry toluene for 15 min. Then, triphenyl phosphine (816 mg, 3.11 mmol) and diethyl azodicarboxylate solution of 40 wt % in toluene (2.57 mL, 5.66 mmol) were added to the ice-cooled solution. After the reaction mixture was stirred at 0°C for an hour, 1.22 mL of DPPA (5.66 mmol) was added dropwise. Then, the reaction mixture was warmed to room temperature and stirred until TLC indicated the disappearance of the starting material. Afterward, the solvent was evaporated in vacuo, affording an orangish oil. Flash chromatography (cyclohexane/ethyl acetate 9:1) was performed to obtain 430 mg (0.90 mmol) of the pure product 36 as a white oil. TLC (cyclohexane/ethyl acetate 9:1); R f = 0.67. 1  1- Azido-1-(1,4-benzodioxan)-propylmethanesulfonate (38). 3-Azido-3-(1,4-benzodioxan)-1-propanol 37 (300 mg, 1.43 mmol) was dissolved in dichloromethane (12 mL), and triethylamine (0.20 mL, 1.68 mmol) was added, and after the reaction mixture was cooled down to 0°C, mesyl chloride (0.13 mL, 1.68 mmol) was added dropwise. The solution was stirred at room temperature until TLC indicated the disappearance of the starting material. The reaction mixture was washed with a 10% aqueous solution of HCl and then with brine. The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was evaporated in vacuo, providing the pure product 38 as 413 mg of a yellow oil (1.32 mmol). TLC (dichloromethane/methanol 95:5); R f = 0.78. 1  4-Hydroxybenzenesulfonamide (39). At 0°C, 5 g of sulfanilamide (29 mmol) was dissolved in water (40 mL) and concentrated sulfuric acid (20 mL). Then, a solution of sodium nitrite (2 g, 29 mmol) in 20 mL of water was added dropwise, and the reaction was stirred at reflux until the evolution of nitrogen ceased. The mixture was cooled down to room temperature and kept overnight at 4°C. Afterward, crystals of 39 (3.78 g, 21.83 mmol) were collected by filtration. Mp 175°C. N,N-Dimethylaminomethylene-4-hydroxybenzenesulfonamide (40). N,N-Dimethylformamide dimethyl acetal (3.87 mL, 29.12 mmol) was added to a solution of 4-hydroxybenzenesulfonamide 39 (4.20 g, 24.27 mmol) in dimethylformamide (4 mL) at 0°C, and the mixture was stirred for 2 h at room temperature. The reaction mixture was treated with ethyl acetate, and the obtained white solid was filtered to afford the title compound 40 (5.21 g, 22.82 mmol). Mp 178°C. 1 H NMR (CD 3 OD): δ 8.12 (s).
CA Inhibition. An Applied Photophysics stopped-flow instrument was used to assay the CA-catalyzed CO 2 hydration activity. 36 Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at an absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as a buffer, and 20 mM Na 2 SO 4 (to maintain constant ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10−100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. 42 Enzyme concentrations ranged between 5 and 12 nM. For each inhibitor, at least six traces of the initial 5−10% of the reaction were used to determine the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the inhibitor (0.1 mM) were prepared in distilled−deionized water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay to allow for the formation of the E−I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng−Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant proteins obtained in-house, as reported earlier. 43−45 Computational Methods. The crystal of human DPP IV was retrieved from RCSB PDB (entry code: 1X70) 46 in complex with ligand 715 (sitagliptin), and the crystal of human CA II was likewise retrieved from RCSB PDB (entry code: 3k34) 47 in complex with ligand SUA (a benzenesulfonamide inhibitor). Since DPP IV is a homodimer, the best chain has been evaluated, choosing the one with the lower number of outliers and the best fitting according to section 6 "Fit of the model and data" of the PDB report.
The obtained structures were then minimized using AMMP, as implemented in the VEGA environment. 48 Both the crystals were then prepared, adding eventual missing residues and checking for alternative conformations of the side chains, chirality, trans-peptide bond, and ring interaction. Hydrogen atoms were added to all crystal structures according to the physiological pH, and they underwent an energy minimization using NAMD2, 49 CHARMM22 as a force field, and constraints on the backbone atoms. To prevent the binding site collapse, the respective ligand was inserted into their crystal structure. Moreover, for CA II, the Zn 2+ ion was reintroduced into the structure by overlapping it with the PDB downloaded structure.
Both binding sites were calculated by redocking, and the bound ligands were removed from the structure for docking experiments. The docking simulations were performed by PLANTS, focusing the search on a 10.0 Å radius sphere around the bound ligands. The conformational profile of the considered ligands was explored by Monte Carlo procedures, as described elsewhere. 50 For each ligand, 10 poses were generated and scored by the ChemPLP score with a speed equal to 1. The complexes were finally minimized by keeping fixed all atoms outside a 10 Å radius sphere around the bound ligand. Interactions between the most potent DDP IV inhibitor compounds and the active site of DPP IV were further investigated. Since compounds of the second and third generations are characterized by two chiral centers, molecular docking investigations were performed on all four diastereomers. For the sake of simplicity, we report only the best results. ■ ASSOCIATED CONTENT